本文關(guān)鍵詞： 鯉魚 TLRs受體 基因克隆 poly(I:C) 嗜水氣單胞菌　出處：《山東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：Toll樣受體(TLRs)是重要的模式識別分子,是連接適應(yīng)性免疫和天然免疫的橋梁。TLRs受體可以分為六大亞家族,TLR1家族、TLR3家族、TLR4家族、TLR5家族、TLR7家族、TLR11家族。魚類是目前已知的TLRs種類最多的物種,目前已經(jīng)在魚類中發(fā)現(xiàn)21種TLRs。對魚類TLRs的研究可以為天然免疫的進(jìn)化研究提供基礎(chǔ)數(shù)據(jù)。本研究運(yùn)用PCR技術(shù)和RACE技術(shù)首次從鯉中克隆得到TLR1和TLR21基因的全長。TLR1的cDNA全長為2626 bp,開放閱讀框(ORF)為2166 bp,編碼721個(gè)氨基酸殘基,TLR21的cDNA全長為3604 bp,ORF包含2892 bp,編碼963個(gè)氨基酸殘基,利用SMART軟件對兩者的結(jié)構(gòu)域進(jìn)行分析,發(fā)現(xiàn)其具有典型的TLRs受體結(jié)構(gòu),包含了一個(gè)胞內(nèi)的TIR結(jié)構(gòu)域、一個(gè)跨膜區(qū)、一個(gè)胞外亮氨酸重復(fù)序列結(jié)構(gòu)域。使用BioEdit軟件對不同物種的氨基酸序列進(jìn)行比對,發(fā)現(xiàn)胞外具有天冬酰胺位點(diǎn),推測與馬蹄狀結(jié)構(gòu)形成有關(guān),胞內(nèi)TIR結(jié)構(gòu)域保守性較高。利用I-TASSER軟件在線模擬TLR1和TLR21的三維空間結(jié)構(gòu),證實(shí)了兩者典型的TLRs受體家族特征。使用MEGA6軟件對不同魚類及部分哺乳動(dòng)物構(gòu)建進(jìn)化樹,發(fā)現(xiàn)鯉魚的TLR1屬于TLR1家族成員,與草魚的親緣關(guān)系最近,而TLR21屬于TLR11家族成員,與斑馬魚和大西洋鮭魚的親緣關(guān)系最近。利用實(shí)時(shí)熒光定量PCR技術(shù)檢測鯉TLR1和TLR21在肝臟、脾臟、頭腎、前腸、后腸、鰓、性腺、皮膚、肌肉、口腔上皮和腦等11種組織中表達(dá)分布情況。結(jié)果顯示,TLR1在腦、脾臟、頭腎和性腺中表達(dá)較高,在后腸和口腔上皮細(xì)胞中表達(dá)較低,TLR21在脾臟和頭腎中表達(dá)較高,在皮膚和肝臟中表達(dá)較低。對鯉進(jìn)行嗜水氣單胞菌注射刺激后,TLR1 mRNA的表達(dá)量在前腸、后腸和肝臟中有明顯升高的趨勢,其中在后腸和肝臟中升高為對照組的7倍,TLR21 mRNA的表達(dá)量在肝臟、前腸和后腸中有升高的趨勢。對鯉進(jìn)行腹腔注射poly(I:C),誘導(dǎo)了其肝臟、脾臟、頭腎、前腸和后腸中TLR1和TLR21基因的表達(dá),肝臟和前腸中的TLR1 mRNA的表達(dá)量有升高趨勢,分別升高為對照組的3倍和2倍,而TLR21 m RNA的表達(dá)量在肝臟、脾臟、前腸和后腸中有明顯上調(diào)趨勢,肝臟中升高的倍數(shù)最高,可為對照組的20倍。本研究為豐富魚類天然免疫提供了基礎(chǔ)資料,同時(shí)也為鯉疾病發(fā)病機(jī)制的研究和防治提供了理論基礎(chǔ)。
[Abstract]:TLRs receptor is an important pattern recognition molecule. It is a bridge between adaptive immunity and innate immunity. TLRs receptor can be divided into six subfamilies: TLR1 family. TLR3 family, TLR4 family, TLR5 family, TLR7 family, TLR11 family. Fish are the most known species of TLRs. At present, 21 species of TLRs have been found in fish. The study on fish TLRs can provide the basic data for the evolutionary study of innate immunity. This study used PCR and RACE technology for the first time from carp. The total length of cDNA of TLR1 and TLR21 gene. TLR1 is 2626. Bp. Open reading frame (ORF) was 2166bp, and the cDNA encoding 721 amino acid residues TLR21 was 3604 BP ORF containing 2892bp. Encoding 963 amino acid residues, SMART software was used to analyze their domains. It was found that they had a typical TLRs receptor structure, including an intracellular TIR domain. A transmembrane domain and an extracellular leucine repeat domain were used to compare the amino acid sequences of different species using BioEdit software. It is speculated that the formation of horseshoe structure is related to the formation of horseshoe structure, and the conserved domain of intracellular TIR is high. I TASSER software is used to simulate the 3D spatial structure of TLR1 and TLR21 on line. The phylogenetic tree of different fish and some mammals were constructed by using MEGA6 software. It was found that the TLR1 of carp belongs to the TLR1 family. TLR21 is a member of the TLR11 family. Real-time fluorescence quantitative PCR was used to detect TLR1 and TLR21 in liver, spleen, head and kidney, foregut, hindgut, Gill, gonad and skin. The results showed that the expression of TLR1 was higher in brain, spleen, head and kidney, and gonad, but lower in hindgut and oral epithelial cells. The expression of TLR21 was higher in spleen and head kidney than in skin and liver. The expression of TLR1 mRNA was found in foregut after injection of Aeromonas hydrophila. The expression of TLR21 mRNA in the hindgut and liver was 7 times higher than that in the control group. The expression of TLR1 and TLR21 genes in liver, spleen, head and kidney, foregut and hindgut of common carp were induced by intraperitoneal injection of polyI: Con. The expression of TLR1 mRNA in liver and foregut was 3 times and 2 times higher than that in control group, while the expression of TLR21 mRNA was found in liver and spleen. There was a significant upward trend in the foregut and hindgut, and the highest increase in liver was 20 times higher than that in the control group. This study provided basic information for enriching the innate immunity of fish. It also provides a theoretical basis for the study and prevention of common carp disease.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S943

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本文編號：1486303